Chromatography Forum

Anyone know of a HPLC method for the analysis of peptides conjugated with KLH? Since the KLH is such a large protein, I am having difficulty eluting it on any of the the columns I have tried. I have tried 100A and 300A, 150mm and 50mm, and CN, Phenyl, C8, C18 and C4 columns.
I am running a slow gradient 95:5 -5:95 in 90 minutes using 0.1% TFA in water and 0.1% TFA in acetonitrile.
So far no luck, any suggestions?

All of the column materials you list are variants for reversed-phase chromatography. Reversed-phase chromatography is not useful for intact proteins in general, as you have discovered in this particular case. If your peptide is highly charged, then use ion-exchange chromatography. If it has significant hydrophobic character, then use hydrophobic interaction chromatography (HIC). HIC has selectivity similar to that of reversed-phase but is compatible with intact proteins. Since KLH is so large (> 300 KDa), it's important to use a column with a pore diameter of at least 1000 Å. If you don't select a column with which the peptide interacts preferentially, then the KLH will dominate the chromatography, binding to the column in an orientation in which the peptide conjugate is not in contact with the stationary phase. The column will then not be able to tell the difference between its presence or absence. If you'd like to discuss this problem further, then I suggest you contact me offlist (aalpert@polylc.com).

you have several things that can go wrong with your separation
and the first that comes to mind reading of the protein is the aggregates
they are huge. are you making sure the protein is in good soluble state?

also can you say the size of peptide itself? does it have anything in its characteristics that can help you in the separation? if yes then you might also consider ion exchange columns

from where do you get the KLH? do you buy it? can you get the purification steps they use for it?
maybe it can help you